1. Field of the Invention
The present invention relates to a method for obtaining a targeted DNA sequence. The sequence can be an entire coding region of a gene. The sequence can also be a template for PCR amplification. The present invention further relates to DNA sequences generated by the method.
2. Description of the Related Art
Isolation of DNA, including genes, may be performed by either classical or molecular genetic approaches. Classical methods are inherently slow and lack the specificity required to identify very small (short) regions of DNA. Molecular approaches for identifying and obtaining a target DNA sequence of interest requires (1) a synthetic DNA primer set designed to recognize the 3' and 5' ends of the target DNA element and subsequent polymerase chain reaction (PCR) amplification or (2) a nucleic acid probe that is complementary to the target DNA for hybridization.
An example of target DNA that is required for biological analysis or procedure is a molecular marker. Molecular markers are associated with any measurable phenotypic or genotypic characteristic. Markers can be used as a source of genetic "fingerprints" and as selectable markers linked to phenotypic traits of interest. Conventional markers used in plant breeding and identification include RFLPS, RAPDs and AFLP.
RFLP analysis is a hybrid of classical and molecular methodology requiring crosses between parents and analysis of parents and siblings for inheritance of a particular nucleic acid element (marker). RAPD and AFLP methods rely upon the presence within genomic DNA of target elements that will be recognized by a selected primer or primer set. These methods are limited by the relatively infrequent positioning of two target elements within a distance that allows amplification of a product of a size that will be useful for analysis by electrophoresis. Restriction fragments used in restriction length polymorphism assays (RFLP) are one example of a molecular marker. RFLP is used, for example, in breeding programs for corn, barley, Brassica (vegetable and oilseed), etc. RFLP markers are of limited utility in crops such as soybean, tomato and wheat where the number of polymorphic RFLP markers are quite small.
Simple Sequence Repeats (SSRs) are another type of marker which are more polymorphic than RFLP markers for a number of plants. SSRs, also called microsatellites, are short segments of DNA consisting of repeated short nucleotide sequences, such as for example, the DNA sequence ATATATAT which is a dinucleotide (AT) repeat with four repeats. The number of nucleotides that repeat generally varies from one to five bases; the number of repeated sequences can range from two to about forty. There are large numbers of repeats that can exist in a genome and the high level of polymorphism that may be associated with repeated sequences make SSRs valuable as molecular markers. The utility of a particular marker is determined by the number of markers available and the polymorphism of those markers. SSRs are found to be both more abundant and more polymorphic than other types of DNA markers. SSRs were first recognized and used by scientists working in human genetics. Most recently, they have been used to develop genome maps of certain animal and plant species. Scientists researching mammalian systems have produced SSR maps of the mouse and bovine genomes and learned that SSRs were well distributed along the chromosomes which is another important criteria for the utility of a molecular marker.
In plants, the use of SSRs can reveal differences among soybean varieties that are indistinguishable using standard RFLP techniques. In certain instances, SSR technology can provide additional markers that can be useful in combination with RFLP procedures. The availability of additional markers increases the probability of finding one or more markers that are tightly linked to a gene or groups of genes controlling a trait of interest. This increased probability can make marker-assisted selection even more efficient.
Conventional SSR development overcomes the limitations of the above mentioned technologies by isolation of marker elements by screening genomic DNA libraries for markers of interest. Using DNA sequence flanking the newly isolated marker (thus obtaining an "allele" for the particular SSR) synthetic oligonucleotide primers are then designed for subsequent PCR analysis of the length of the repeat element at that allele. SSR length varies considerably within alleles and SSR alleles are abundant and ubiquitous throughout the genome of most eukaryotic organisms. However, genomic library preparation requires relatively large amounts of genomic DNA, compared to RAPD or AFLP markers, and is difficult and time consuming. Library screening is also difficult and time consuming, with library preparation and screening requiring several months to complete.
Inverse PCR (Ochman et al., Genetics, Volume 120, 621-623, 1988; Triglia et al., Nucleic Acid Research, Volume 16, 8186, 1988; Silver et al., J. Virol., Volume 63, 1924-1928, 1989) permits amplification of DNA flanking a known sequence by circulization of restriction enzyme digested DNA. This permits amplification of the flanking sequence by positioning two primers, each of which binds to the known sequence "inside out" on the circle. This strategy maintains specificity at each primer binding site. However, difficulties with inverse PCR include the requirement of two restriction sites that flank the priming region and inefficient PCR amplification of closed circular DNA.
Yield is improved in conventional PCR when circular DNA template is linearized. If a restriction site is not present to linearize the circle between the 5' ends of the amplifying primers, the standard PCR amplification is less efficient than reverse PCR. Without linearization, double-stranded circular DNA is amplified much less efficiently than linear DNA (Jones et al., Biotechniques, Volume 10, 62-66, 1991). Nicking the circles, by heating, ameliorates the difficulty in amplifying closed circular double-stranded DNA, but only a small percentage of the circles are nicked between the two 5' ends of the amplifying primers. Any increase in the initial amplification efficiency is suboptimal.
U.S. Pat. No. 5,411,875 (Jones) discloses a PCR method where a single strand oligonucleotide is ligated to restriction enzyme digested DNA. The oligonucleotide is constructed to be complementary to a known region of DNA upstream from the unknown region of DNA. This is then denatured and self-annealed resulting in a single stranded loop or pan portion with a double-strand of an otherwise single-stranded handle. This permits PCR amplification of the unknown DNA using thermostable DNA polymerase having single-stranded 3' exonuclease activity. This method is susceptible to nonspecific hybridization.
There is a need in the art to be able to generate target DNA in less time with greater efficiency. The present invention provides a method, different from the related art, which eliminates genomic DNA library preparation and screening which are the most time consuming steps, typically requiring no less than three months, with total time for target DNA development being between 4-6 months.